Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes

• Published in: The Journal of biological chemistry 1994-12, Vol.269 (48), p.30164-30172
• Main Authors: Quick, M W, Simon, M I, Davidson, N, Lester, H A, Aragay, A M
• Format: Article
• Language: English
• Subjects: Adenylate Cyclase Toxin; Animals; Base Sequence; Bordetella pertussis; Chloride Channels - drug effects; Chloride Channels - physiology; GTP-Binding Proteins - metabolism; Humans; Isoenzymes - antagonists & inhibitors; Isoenzymes - biosynthesis; Kinetics; Macromolecular Substances; Membrane Potentials - drug effects; Membrane Potentials - physiology; Molecular Sequence Data; Oligodeoxyribonucleotides - pharmacology; Oligonucleotides, Antisense - pharmacology; Oocytes; Pertussis Toxin; Receptors, Serotonin - biosynthesis; Receptors, Serotonin - metabolism; Receptors, Thyrotropin-Releasing Hormone - biosynthesis; Receptors, Thyrotropin-Releasing Hormone - metabolism; RNA, Complementary - metabolism; Type C Phospholipases - antagonists & inhibitors; Type C Phospholipases - biosynthesis; Virulence Factors, Bordetella - pharmacology; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)43792-1

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.